Monitoring of antibody genes during an immune response shows that extensive mutation of the Ig genes that respond to the infection takes place in B cells within germinal centers. A direct demonstration that germinal centers are the sites of somatic hypermutation comes from the work of G. Kelsoe and his colleagues. These workers compared the mutation frequencies in B cells isolated from germinal centers with those from areas of intense B-cell activation outside the germinal centers. To do so, they prepared thin sections of spleen tissue from animals immunized with the hapten 4-hydroxy-3-nitrophenylacetyl (NP) conjugated with chicken gamma globulin as a carrier. This system is convenient because the initial response to this hapten is dominated by a particular heavy-chain gene rearrangement and the use of a 7 light chain (in mice, >95% of antibodies bear k light chains). Consequently, antibodies against the idiotype of this antibody can be used to readily distinguish responding B cells. Using antibodies to the idiotype and immunohistological staining techniques, these workers identified B cells bearing anti-NP antibody in germinal centers and nongerminal-center foci of B-cell activation present in thin sections cut from the spleens of recently immunized mice. They isolated these B cells by microdissection, used PCR to amplify the immunoglobulin genes of each individual cell, and then cloned and sequenced the immunoglobulin genes. Many mutations were found in the immunoglobulin genes obtained from B cells in germinal centers, few in the genes obtained from activated B cells in nongerminal-center foci. When the mutated sequences of the collection of B cells from germinal centers was examined, it was apparent that many of the cells had sequences that were sufficiently similar that they were likely to be related by common descent from the same precursor cell. Detailed analysis of the sequences allowed these workers to build genealogic trees in which one could clearly see the descent of progeny from progenitors by progressive somatic hypermutation.
The introduction of point mutations, deletions, and insertions into the rearranged immunoglobulin genes is strikingly focused. Figure 11-18 shows that the overwhelming majority of these mutations occur in a region that extends from about 0.5 kb 5' to about 1.5 kb 3' of the V(D)J segments of rearranged immunoglobulin genes. Although the hypermutation process delivers mutations throughout the V region, antigen-driven selection results in the eventual emergence of immunoglobulin genes in which the majority of the mutations lie within the three complementarity-determining regions (CDRs). It has been estimated that the mutation rate during somatic mutation is approximately 10-3/base pair/ division, which is a millionfold greater than the normal mutation rate for other genes of humans or mice cells. Since the heavy- and light-chain V(D)J segments total about 700 base pairs, this rate of mutation means that, for every two cell divisions it undergoes, a centroblast will acquire a mutation in either the heavy- or light-chain variable regions. The extremely high rates and precise targeting of somatic hypermutation are remarkable features that are unique to the immune system. Determining the molecular basis of this extraordinary process remains a challenge in immunology.
Because somatic mutation occurs randomly, it will generate a few cells with receptors of higher affinity and many cells with receptors of unchanged or lower affinity for a particular antigen. Therefore, selection is needed to derive a population of cells that has increased affinity. The germinal center is the site of selection. B cells that have high-affinity receptors for the antigen are likely to be positively selected and leave the germinal center; those with low affinity are likely to undergo negative selection and die in the germinal center.
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